This project is designed to determine the important molecular events that control T cell development within the thymus. After migrating to the thymus, hematopoietic precursor cells undergo a complex set of differentiation events and emerge as mature T lymphocytes capable of specific antigen recognition. This developmental process appears to result from a series of signalling interactions between T cell precursors and a heterogeneous and as yet incompletely-defined set of stromal cells. We have made progress in three areas of thymocyte differentiation: First, we have established a culture system in which day 14/15 triple negative fetal thymocytes can be stimulated to express early markers of thymocyte development including CD25, ICAM-1, and Ly-6A/E. We have found that IL-1 and tumor necrosis factor are critical mediators of thymic development. We have also studied precursor populations in the thymus that gives rise to T cells, B cells, and natural killer cells and have determined that the NK1.1 marker identifies a very early progenitor population whose developmental potential is restricted to the T and natural killer lymphocyte lineages. Second, we have discovered that the block to the development of double positive T lymphocytes in mice deficient for the recombinase-activating gene (RAG) can be overcome by gamma-irradiation. We have found that this involves somatic mutation of the p53 locus leading to the production of mutant p53 protein. These data suggest that there is a p53-mediated checkpoint in early thymocyte development. Third, we have studied the molecular mechanism of programmed cell death during thymocyte maturation. Programmed cell death acts in the thymus to eliminate thymocytes which bear antigen receptors that are incapable of recognizing self-MHC molecules (positive selection) or that react with self proteins too strongly (negative selection). We have found that both events involve the activation of a class of cysteine proteases (termed caspases) that have also been implicated in the programmed death of mature T lymphocytes as well as other somatic cells. We are currently exploring the molecular pathways by which these enzymes are activated and lead to programmed death. These studies promise to shed light on a key regulatory event that ensures proper antigen recognition and avoids self-reactivity that could lead to autoimmune diseases.